Caspase inhibitors and uses thereof

ABSTRACT

This invention provides caspase inhibitors of formula I: 
                         
wherein Z is oxygen or sulfur; R 1  is hydrogen, —CHN 2 , R, CH 2 OR, CH 2 SR, or —CH 2 Y;   between R 3  and R 4  represents a single or double bond; Y is an electronegative leaving group; R 2  is CO 2 H, CH 2 CO 2 H, or esters, amides or isosteres thereof; R 3  is a group capable of fitting into the S2 subsite of a caspase enzyme; R 4  is a hydrogen or C 1-6  alkyl or R 3  and R 4  taken together form a ring; Ring A and Ring B are each heterocyclic rings, and R and R 5  are as described in the specification. The compounds are effective inhibitors of apoptosis and IL-1β secretion.

CROSS REFERENCE TO RELATED APPLICATIONS

Pursuant to Title 35, United States Code, 119 and §120, this applicationis a divisional of U.S. patent application Ser. No. 11/770,093, filedJun. 28, 2007, which is a divisional of U.S. patent application Ser. No.10/153,971, filed May 23, 2002, now U.S. Pat. No. 7,351,702; whichclaims the benefit of U.S. Provisional Application 60/292,969, filed May23, 2001, the contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

This invention is in the field of medicinal chemistry and relates tonovel compounds, and pharmaceutical compositions thereof, that inhibitcaspases that mediate cell apoptosis and inflammation. The inventionalso relates to methods of using the compounds and pharmaceuticalcompositions of this invention to treat diseases where caspase activityis implicated.

BACKGROUND OF THE INVENTION

Apoptosis, or programmed cell death, is a principal mechanism by whichorganisms eliminate unwanted cells. The deregulation of apoptosis,either excessive apoptosis or the failure to undergo it, has beenimplicated in a number of diseases such as cancer, acute inflammatoryand autoimmune disorders, ischemic diseases and certainneurodegenerative disorders (see generally Science, 1998, 281,1283-1312; Ellis et al., Ann. Rev. Cell. Biol., 1991, 7, 663).

Caspases are a family of cysteine protease enzymes that are keymediators in the signaling pathways for apoptosis and cell disassembly(Thornberry, Chem. Biol., 1998, 5, R97-R103). These signaling pathwaysvary depending on cell type and stimulus, but all apoptosis pathwaysappear to converge at a common effector pathway leading to proteolysisof key proteins. Caspases are involved in both the effector phase of thesignaling pathway and further upstream at its initiation. The upstreamcaspases involved in initiation events become activated and in turnactivate other caspases that are involved in the later phases ofapoptosis.

Caspase-1, the first identified caspase, is also known as interleukinconverting enzyme or “ICE.” Caspase-1 converts precursor interleukin-1β(“pIL-1β”) to the pro-inflammatory active form by specific cleavage ofpIL-1β between Asp-116 and Ala-117. Besides caspase-1 there are alsoeleven other known human caspases, all of which cleave specifically ataspartyl residues. They are also observed to have stringent requirementsfor at least four amino acid residues on the N-terminal side of thecleavage site.

The caspases have been classified into three groups depending on theamino acid sequence that is preferred or primarily recognized. The groupof caspases, which includes caspases 1, 4, and 5, has been shown toprefer hydrophobic aromatic amino acids at position 4 on the N-terminalside of the cleavage site. Another group which includes caspases 2, 3and 7, recognize aspartyl residues at both positions 1 and 4 on theN-terminal side of the cleavage site, and preferably a sequence ofAsp-Glu-X-Asp. A third group, which includes caspases 6, 8, 9 and 10,tolerate many amino acids in the primary recognition sequence, but seemto prefer residues with branched, aliphatic side chains such as valineand leucine at position 4.

The caspases have also been grouped according to their perceivedfunction. The first subfamily consists of caspases-1 (ICE), 4, and 5.These caspases have been shown to be involved in pro-inflammatorycytokine processing and therefore play an important role ininflammation. Caspase-1, the most studied enzyme of this class,activates the IL-1β precursor by proteolytic cleavage. This enzymetherefore plays a key role in the inflammatory response. Caspase-1 isalso involved in the processing of interferon gamma inducing factor(IGIF or IL-18) which stimulates the production of interferon gamma, akey immunoregulator that modulates antigen presentation, T-cellactivation and cell adhesion.

The remaining caspases make up the second and third subfamilies. Theseenzymes are of central importance in the intracellular signalingpathways leading to apoptosis. One subfamily consists of the enzymesinvolved in initiating events in the apoptotic pathway, includingtransduction of signals from the plasma membrane. Members of thissubfamily include caspases-2, 8, 9 and 10. The other subfamily,consisting of the effector caspases 3, 6 and 7, are involved in thefinal downstream cleavage events that result in the systematic breakdownand death of the cell by apoptosis. Caspases involved in the upstreamsignal transduction activate the downstream caspases, which then disableDNA repair mechanisms, fragment DNA, dismantle the cell cytoskeleton andfinally fragment the cell.

A four amino acid sequence primarily recognized by the caspases has beendetermined for enzyme substrates. Talanian et al., J. Biol. Chem. 272,9677-9682, (1997); Thornberry et al., J. Biol. Chem. 272, 17907-17911,(1997). Knowledge of the four amino acid sequence primarily recognizedby the caspases has been used to design caspase inhibitors. Reversibletetrapeptide inhibitors have been prepared having the structureCH₃CO—[P4]-[P3]-[P2]—CH(R)CH₂CO₂H where P2 to P4 represent an optimalamino acid recognition sequence and R is an aldehyde, nitrile or ketonecapable of binding to the caspase cysteine sulfhydryl. Rano andThornberry, Chem. Biol. 4, 149-155 (1997); Mjalli, et al., Bioorg. Med.Chem. Lett. 3, 2689-2692 (1993); Nicholson et al., Nature 376, 37-43(1995). Irreversible inhibitors based on the analogous tetrapeptiderecognition sequence have been prepared where R is anacyloxymethylketone —COCH₂OCOR′. R′ is exemplified by an optionallysubstituted phenyl such as 2,6-dichlorobenzoyloxy and where R is COCH₂Xwhere X is a leaving group such as F or Cl. Thornberry et al.,Biochemistry 33, 3934 (1994); Dolle et al., J Med. Chem. 37, 563-564(1994).

The utility of caspase inhibitors to treat a variety of mammaliandisease states associated with an increase in cellular apoptosis hasbeen demonstrated using peptidic caspase inhibitors. For example, inrodent models, caspase inhibitors have been shown to reduce infarct sizeand inhibit cardiomyocyte apoptosis after myocardial infarction, toreduce lesion volume and neurological deficit resulting from stroke, toreduce post-traumatic apoptosis and neurological deficit in traumaticbrain injury, to be effective in treating fulminant liver destruction,and to improve survival after endotoxic shock. Yaoita et al.,Circulation, 97, 276 (1998); Endres et al., J Cerebral Blood Flow andMetabolism, 18, 238, (1998); Cheng et al., J. Clin. Invest., 101, 1992(1998); Yakovlev et al., J Neuroscience, 17, 7415 (1997); Rodriquez etal., J. Exp. Med., 184, 2067 (1996); Grobmyer et al., Mol. Med., 5, 585(1999).

In general, the peptidic inhibitors described above are very potentagainst some of the caspase enzymes. However, this potency has notalways been reflected in cellular models of apoptosis. In additionpeptide inhibitors are typically characterized by undesirablepharmacological properties such as poor oral absorption, poor stabilityand rapid metabolism. Plattner and Norbeck, in Drug DiscoveryTechnologies, Clark and Moos, Eds. (Ellis Horwood, Chichester, England,1990).

There are reports of modified peptide inhibitors. WO 91/15577 and WO93/05071 disclose peptide ICE inhibitors of the formula:Z-Q₂-Asp-Q₁wherein Z is an N-terminal protecting group; Q₂ is 0 to 4 amino acids;and Q₁ is an electronegative leaving group.

WO 99/18781 discloses dipeptide caspase inhibitors of the formula:

wherein R₁ is an N-terminal protecting group; AA is a residue of anatural α-amino acid or β-amino acid; R₂ is hydrogen or CH₂R₄ where R₄is an electronegative leaving group; and R₃ is alkyl or hydrogen.

WO 99/47154 discloses dipeptide caspase inhibitors of the formula:

wherein R₁ is an N-terminal protecting group; AA is a residue of anon-natural α-amino acid or β-amino acid; and R₂ is optionallysubstituted alkyl or hydrogen.

WO 00/023421 discloses (substituted) acyl dipeptide apoptosis inhibitorshaving the formula:

where n is 0, 1, or 2; q is 1 or 2; A is a residue of certain natural ornon-natural amino acid; B is a hydrogen atom, a deuterium atom, C₁₋₁₀straight chain or branched alkyl, cycloalkyl, phenyl, substitutedphentyl, naphthyl, substituted naphthyl, 2-benzoxazolyl, substituted2-oxazolyl, (CH₂)_(m)cycloalkyl, (CH₂)_(m)phenyl, (CH₂)_(m)(substitutedphenyl), (CH₂)_(m)(1- or 2-naphthyl), (CH₂)_(m)heteroaryl, halomethyl,CO₂R¹³, CONR¹⁴R¹⁵, CH₂ZR¹⁶, CH₂OCOaryl, CH₂OCO(substituted aryl),CH₂OCO(heteroaryl), CH₂OCO(substituted heteroaryl), or CH₂OPO(R¹⁷)R¹⁸,where R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷ and R¹⁸ are defined in the application; R²is selected from a group containing hydrogen, alkyl, cycloalkyl, phenyl,substituted phenyl, (CH₂)_(m)NH₂; R³ is hydrogen, alkyl, cycloalkyl,(cycloalkyl)alkyl, phenylalkyl, or substituted phenylalkyl; X is CH₂,C═O, O, S, NH, C═ONH or CH₂OCONH; and Z is an oxygen or a sulfur atom.

WO 97/24339 discloses inhibitors of interleukin-1β converter enzyme ofthe formula:

wherein R¹ represents H, alkyl, alkoxy, a carbocycle, a heterocycle, andvarious other groups; AA¹ and AA² are single bonds or amino acids; and Yrepresents a group of formula:

wherein the Tet ring represents a tetrazole ring; and Z represents,inter alia, alkylene, alkenylene, O, S, SO, and SO₂.

EP 618223 discloses ICE inhibitors of the formula:R-A₁-A₂-X-A₃wherein R is H, a protecting group, or an optionally ring substitutedPhCH₂O; A₁ is an α-hydroxy- or α-amino acid residue; A₂ is anα-hydroxyacid residue or α-amino acid or A₁ and A₂ form together apseudodipeptide or a dipeptide mimetic residue; X is a residue derivedfrom Asp wherein A₃ is CH₂X₁COY₁, CH₂OY₂, CH₂SY₃ or CH₂(CO)_(m)Y₆wherein X₁ is O or S, m is 0 or 1 and Y₁, Y₂, Y₃ and Y₆ are optionallysubstituted cyclic aliphatic or aryl groups.

WO 98/16502 discloses, inter alia, ICE inhibitors of the formula:

wherein R₁ and R₂ are as described in the application and thepyrrolidine ring is substituted by various groups.

WO 99/56765 discloses ICE inhibitors of the formula:

wherein R¹, R³, R⁴ and Y are described in the application and R¹ and R²are independently hydrogen, C₁-C₆ alkyl, OH, (CH₂)_(n)-substituted aryl,(CH₂)_(n)—O-aryl, (CH₂)_(n)—O-substituted aryl, (CH₂)_(n)—S-aryl,(CH₂)_(n)—S-substituted aryl, (CH₂)_(n)—S-heteroaryl,(CH₂)_(n)—S-substituted heteroaryl, (CH₂)_(n)—NR′-aryl,(CH₂)_(n)—NR′-substituted aryl, (CH₂)_(n)—NR′-heteroaryl,(CH₂)_(n)—NR′-substituted heteroaryl, (CH₂)_(n)-heteroaryl,(CH₂)_(n)-substituted heteroaryl, each n is independently 0-6.

While a number of caspase inhibitors have been reported, it is not clearwhether they possess the appropriate pharmacological properties to betherapeutically useful. Therefore, there is a continued need for smallmolecule caspase inhibitors that are potent, stable, and penetratemembranes to provide effective inhibition of apoptosis in vivo. Suchcompounds would be extremely useful in treating the aforementioneddiseases where caspase enzymes play a role.

SUMMARY OF THE INVENTION

It has now been found that compounds of this invention andpharmaceutical compositions thereof are effective as inhibitors ofcaspases and cellular apoptosis. These compounds have the generalformula I:

wherein:

-    between R³ and R⁴ represents a single or double bond;-   Z is oxygen or sulfur;-   R¹ is hydrogen, —CHN₂, —R, —CH₂OR, —CH₂SR, or —CH₂Y;-   R is a C₁₋₁₂ aliphatic, aryl, aralkyl, heterocyclyl, or    heterocyclylalkyl;-   Y is an electronegative leaving group;-   R² is CO₂H, CH₂CO₂H, or esters, amides or isosteres thereof;-   R³ is a group capable of fitting into the S2 sub-site of a caspase;-   R⁴ is hydrogen or a C₁₋₆ aliphatic group that is optionally    interrupted by —O—, —S—, —SO₂—, —CO—, —NH—, or —N(C₁₋₄ alkyl)-, or    R³ and R⁴ taken together with their intervening atoms optionally    form a 3-7 membered ring having 0-2 heteroatoms selected from    nitrogen, oxygen or sulfur;-   Ring A is a nitrogen-containing mono-, bi- or tricyclic ring system    having 0-5 additional ring heteroatoms selected from nitrogen,    oxygen or sulfur;-   Ring B is a nitrogen-containing 5-7 membered ring having 0-2    additional ring heteroatoms selected from nitrogen, oxygen or    sulfur;-   R⁵ is R⁶, (CH₂)_(n)R⁶, COR⁶, CO₂R⁶, SO₂R⁶, CON(R⁶)₂, or SO₂N(R⁶)₂;-   n is one to three; and-   each R⁶ is independently selected from hydrogen, an optionally    substituted C₁₋₄ aliphatic group, an optionally substituted C₆₋₁₀    aryl group, or a mono- or bicyclic heteroaryl group having 5-10 ring    atoms.

The compounds of this invention have inhibition properties across arange of caspase targets with good efficacy in cellular models ofapoptosis. In addition, these compounds will have good cell penetrationand pharmacokinetic properties and, as a consequence of their potency,have good efficacy against diseases where caspases are implicated.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides novel compounds, and pharmaceutically acceptablederivatives thereof, that are useful as caspase inhibitors. Theinvention also provides methods for using the compounds to inhibitcaspase activity and to treat caspase-mediated diseases. These compoundshave the general formula I:

wherein:

-    between R³ and R⁴ represents a single or double bond;-   Z is oxygen or sulfur;-   R¹ is hydrogen, —CHN₂, —R, —CH₂OR, —CH₂SR, or —CH₂Y;-   R is a C₁₋₁₂ aliphatic, aryl, aralkyl, heterocyclyl, or    heterocyclylalkyl;-   Y is an electronegative leaving group;-   R² is CO₂H, CH₂CO₂H, or esters, amides or isosteres thereof;-   R³ is a group capable of fitting into the S2 sub-site of a caspase;-   R⁴ is hydrogen or a C₁₋₆ aliphatic group that is optionally    interrupted by —O—, —S—, SO₂—, —CO—, —NH—, or —N(C₁₋₄ alkyl)-, or R³    and R⁴ taken together with their intervening atoms optionally form a    3-7 membered ring having 0-2 heteroatoms selected from nitrogen,    oxygen or sulfur;-   Ring A is a nitrogen-containing mono-, bi- or tricyclic ring system    having 0-5 additional ring heteroatoms selected from nitrogen,    oxygen or sulfur;-   Ring B is a nitrogen-containing 5-7 membered ring having 0-2    additional ring heteroatoms selected from nitrogen, oxygen or    sulfur;-   R⁵ is R⁶, (CH₂)_(n)R, COR⁶, CO₂R⁶, SO₂R⁶, CON(R⁶)₂, or SO₂N(R⁶)₂;-   n is one to three; and-   each R⁶ is independently selected from hydrogen, an optionally    substituted C₁₋₄ aliphatic group, an optionally substituted C₆₋₁₀    aryl group, or a mono- or bicyclic heteroaryl group having 5-10 ring    atoms.

As used herein, the following definitions shall apply unless otherwiseindicated. The term “aliphatic” as used herein means straight chained orbranched C₁-C₁₂ hydrocarbons which are completely saturated or whichcontain one or more units of unsaturation. Aliphatic groups includesubstituted or unsubstituted linear, branched or cyclic alkyl, alkenyl,or alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl,(cycloalkenyl)alkyl or (cycloalkyl)alkenyl. The term “alkyl” used aloneor as part of a larger moiety refers to both straight and branchedchains containing one to twelve carbon atoms. When the term alkyl isused as part of a larger moiety, as in aralkyl or heteroaralkyl, thealkyl portion will preferably contain one to six carbons.

The term “halogen” means F, Cl, Br, or I. The term “aryl” refers tomonocyclic or polycyclic aromatic ring groups having five to fourteenatoms, such as phenyl, naphthyl and anthryl.

The term “heterocyclic group” refers to saturated and unsaturatedmonocyclic or polycyclic ring systems containing one or more heteroatomsand a ring size of three to nine such as furanyl, thienyl, pyrrolyl,pyrrolinyl, pyrrolidinyl, dioxolanyl, oxazolyl, thiazolyl, imidazolyl,imidazolinyl, imidazolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl,isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyranyl,pyridinyl, piperidinyl, dioxanyl, morpholinyl, dithianyl,thiomorpholinyl, pyridazinyl, pyrimidinyl, pyrazinyl, piperazinyl,triazinyl, trithianyl, indolizinyl, indolyl, isoindolyl, indolinyl,benzofuranyl, benzothiophenyl, indazolyl, benzimidazolyl, benzthiazolyl,purinyl, quinolizinyl, quinolinyl, isoquinolinyl, cinnolinyl,phthalazinyl, quinazolinyl, quinoxalinyl, 1,8-naphthyridinyl,pteridinyl, quinuclidinyl, carbazolyl, acridinyl, phenazinyl,phenothiazinyl, or phenoxazinyl. “Heteroaryl” refers to a heterocyclicring that is aromatic. It is understood that the compounds of thisinvention are limited to those that can exist in nature as stablechemical compounds.

The term “carbocyclic group” refers to saturated monocyclic orpolycyclic carbon ring systems of three to fourteen carbons which may befused to aryl or heterocyclic groups. Examples include cyclohexyl,cyclopentyl, cyclobutyl, cyclopropyl, indanyl, tetrahydronaphthyl andthe like.

The terms aliphatic, alkyl, aryl, heteroaryl, heterocyclyl, orcarbocyclyl, used alone or as part of a larger moiety, refers tosubstituted or unsubstituted groups. When substituted, these groups maycontain one or more substituents. Examples of suitable substituentsinclude halogen, —R, —OR, —OH, —SH, —SR, protected OH (such as acyloxy),phenyl (Ph), substituted Ph, —OPh, substituted —OPh, —NO₂, —CN, —NH₂,—NHR, —N(R)₂, —NHCOR, —NHCONHR, —NHCON(R)₂, —NRCOR, —NHCO₂R, —CO₂R,—CO₂H, —COR, —CONHR, —CON(R)₂, —S(O)₂R, —SONH₂, —S(O)R, —SO₂NHR,—NHS(O)₂R, ═O, ═S, ═NNHR, ═NNR₂, ═N—OR, ═NNHCOR, ═NNHCO₂R, ═NNHSO₂R, or═NR where R is an aliphatic group or a substituted aliphatic grouppreferably having one to six carbons, more preferably having one to fourcarbons.

A substitutable nitrogen on a heterocyclic ring may be optionallysubstituted. Suitable substituents on the nitrogen include R, COR,S(O)₂R, and CO₂R, where R is an aliphatic group or a substitutedaliphatic group preferably having one to six carbons, more preferablyhaving one to four carbons.

Nitrogen and sulfur may be in their oxidized form, and nitrogen may bein a quaternized form.

The term “electronegative leaving group” has the definition known tothose skilled in the art (see March, Advanced Organic Chemistry, 4^(th)Edition, John Wiley & Sons, 1992). Examples of electronegative leavinggroups include halogens such as F, Cl, Br, I, aryl, and alkylsulfonyloxygroups, trifluoromethanesulfonyloxy, OR, SR, —OC═O(R), —OPO(R⁷)(R⁸),where R is an aliphatic group, an aryl group, an aralkyl group, aheterocyclic group, or a heterocyclylalkyl group; and R⁷ and R⁸ areindependently selected from R or OR.

When the R² group is in the form of an ester or amide, the presentcompounds undergo metabolic cleavage to the corresponding carboxylicacids, which are the active caspase inhibitors. Because they undergometabolic cleavage, the precise nature of the ester or amide group isnot critical to the working of this invention. The structure of the R²group may range from the relatively simple diethyl amide to a steroidalester. Examples of esters of R² carboxylic acids include, but are notlimited to, C₁₋₁₂ aliphatic, such as C₁₋₆ alkyl or C₃₋₁₀ cycloalkyl,aryl, such as phenyl, aralkyl, such as benzyl or phenethyl, heterocyclylor heterocyclylalkyl. Examples of suitable R² heterocyclyl ringsinclude, but are not limited to, 5-6 membered heterocyclic rings havingone or two heteroatoms such as piperidinyl, piperazinyl, or morpholinyl.

Amides of R² carboxylic acids may be primary, secondary or tertiary.Suitable substituents on the amide nitrogen include, but are not limitedto, one or more groups independently selected from the aliphatic, aryl,aralkyl, heterocyclyl or heterocyclylalkyl groups described above forthe R² ester alcohol. Likewise, other prodrugs are included within thescope of this invention. See generally Bradley D. Anderson, “Prodrugsfor Improved CNS Delivery” in Advanced Drug Delivery Reviews (1996), 19,171-202.

Isosteres or bioisosteres of R² carboxylic acids, esters and amidesresult from the exchange of an atom or group of atoms to create a newcompound with similar biological properties to the parent carboxylicacid or ester. The bioisosteric replacement may be physicochemically ortopologically based. An example of an isosteric replacement for acarboxylic acid is CONHSO₂(alkyl) such as CONHSO₂Me.

R³ may be any group capable of fitting into the S2 sub-site of a knowncaspase. Such groups are known from the many caspase inhibitors thathave been reported (see WO91/15577, WO93/05071, WO99/18781, WO99/47154,WO00/023421, WO9724339, EP618223, WO9816502, all of which are describedabove). Furthermore, the structures of several of the caspase enzymesincluding their S-2 subsites are also known. References to the caspasestructure include the following: Blanchard H, et al., J. Mol. Biol.302(1), 9-16 (2000); Wei Y, et al., Chem. Biol. 7(6):423-32 (2000); LeeD, et al., J Biol. Chem. 275(21):16007-14 (2000); Blanchard H, et al.,Structure Fold Des. 7(9):1125-33 (1999); Okamoto Y, et al, Chem. Pharm.Bull. (Tokyo) 47(1):11-21 (1999); Margolin N, et al, J. Biol. Chem.272(11):7223-8 (1997); Walker N P, et al., Cell 78(2):343-52 (1994); andWilson K P, et al., Nature 370(6487):270-5 (1994).

Whether a group will fit into the S-2 subsite will depend on theparticular caspase that is being considered. The size of the subsitewill range from the small S-2 subsite of caspase-3 which permits a groupup to the size of a C₄ aliphatic group to a relatively large subsitewhich permits a group having a molecular weight up to about 140 Daltons,such as a naphthyl group. The size, along with the electronic nature, ofthe R³ group will influence the caspase selectivity of the inhibitor.From the references provided above, one skilled in the art could readilyascertain whether a group is capable of fitting favorably into an S-2subsite of a caspase, for example, by using standard molecular modelingprograms such as Quanta or Macromodel.

Suitable R³ groups include hydrogen, a side chain of a natural α-aminoacid, or a substituted or unsubstituted group having a molecular weightup to about 140 Daltons selected from aliphatic, aryl, aralkyl,heterocyclyl, and heterocyclylalkyl groups. Examples of R³ aliphaticgroups include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl,isobutyl, sec-butyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl,and cyclohexyl. Examples of R³ aryl groups include phenyl, indenyl andnaphthyl. Examples of R³ heterocyclic groups include pyrrolidinyl,pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolinyl, pyrazolidinyl,piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, homopiperidinyl,and quinuclidinyl. Examples of R³ heteroaryl groups include furanyl,thienyl, pyrrolyl, oxazole, thiazolyl, imidazolyl, pyrazolyl,isoxazolyl, isothiazolyl, oxadiazolyl, furazanyl, triazolyl,thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl,indolyl, isoindolyl, indolinyl, benzofuranyl, benzothiophene, indazolyl,benzimidazolyl, benzthiazolyl, purinyl, quinolinyl, isoquinolinyl,quinolizinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl,naphthyridinyl, pteridinyl, chromanyl, and isochromanyl. Each group maycontain one or more substituents, as described above.

Ring A is an optionally substituted nitrogen-containing mono-, bi- ortricyclic ring system having 0-5 additional ring heteroatoms selectedfrom nitrogen, oxygen or sulfur, preferably having 0-3 additional ringheteroatoms. Such rings include substituted or unsubstituted indole,isoindole, indoline, indazole, purine, dihydropyridine, benzimidazole,imidazole, imidazoline, pyrrole, pyrrolidine, pyrroline, pyrazole,pyrazoline, pyrazolidine, triazole, piperidine, morpholine,thiomorpholine, piperazine, carbazole, iminostilbene, phenothiazine,phenoxazine, dihydrophenazine, dihydrocinnoline, dihydroquinoxaline,tetrahydroquinoline, tetrahydroisoquinoline, dihydronaphthyridine,tetrahydronaphthyridine, dihydroacridine, 5H-dibenzo[b,f]azepine,10,11-dihydro-5H-dibenzo[b,f]azepine, β-carboline, pyrido[4,3-b]indole,2,3,9-triazafluorene, 9-thia-2,10-diazaanthracene, 3,6,9-triazafluorene,thieno[3,2-b]pyrrole, or dihydrophenanthridine. Suitable substituents onRing A include one or more groups independently selected from a halogen,—R, —OR, —OH, —SH, —SR, protected OH (such as acyloxy), phenyl (Ph),substituted Ph, —OPh, substituted —OPh, —NO₂, —CN, —NH₂, —NHR, —N(R)₂,—NHCOR, —NHCONHR, —NHCON(R)₂, —NRCOR, —NHCO₂R, —CO₂R, —CO₂H, —COR,—CONHR, —CON(R)₂, —S(O)₂R, —SONH₂, —S(O)R, —SO₂NHR, or —NHS(O)₂R, whereeach R is independently selected from an aliphatic group or asubstituted aliphatic group. The R groups preferably have one to sixcarbons, more preferably one to four carbons.

Compounds of this invention where R² is COOH are gamma-ketoacids, whichmay exist in solution as either the open form 1a or the cyclizedhemiketal form 1a′. The representation herein of either isomeric form ismeant to include the other. Similarly, cyclization may also occur whereR² is CH₂COOH, and such cyclized isomers are understood to be includedwhen the ring open form is represented herein.

Likewise it will be apparent to one skilled in the art that certaincompounds of this invention may exist in tautomeric forms or hydratedforms, all such forms of the compounds being within the scope of theinvention. Unless otherwise stated, structures depicted herein are alsomeant to include all stereochemical forms of the structure; i.e., the Rand S configurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds are within the scope of the invention.Unless otherwise stated, structures depicted herein are also meant toinclude compounds that differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of hydrogen by a deuterium ortritium, or the replacement of a carbon by a ¹³C— or ¹⁴C-enriched carbonare within the scope of this invention.

One embodiment of this invention relates to compounds of formula Ia.Another embodiment relates to compounds of formula Ib. It is preferredthat Z is oxygen. It is also preferred that

 between R³ and R⁴ is a single bond. Having the single bond will providestereoisomers if R³ or R⁴ are other than hydrogen. Preferredstereoisomers of the present compounds will have the followingconfiguration:

Another embodiment of this invention relates to compounds of formula Iathat have one or more, and preferably all, of the following features:

(i) R¹ is hydrogen, —R, —CH₂OR, —CH₂SR, or —CH₂Y. More preferably, R¹ is—CH₂OR, —CH₂SR, or —CH₂Y. An even more preferred R¹ is —CH₂Y. Mostpreferably, R¹ is —CH₂F.

(ii) R² is CO₂H or an ester, amide or isostere thereof.

(iii) R³ is a group having a molecular weight up to about 140 Daltons,such as an aliphatic or aralkyl group. More preferably, R³ is a C₁-C₄alkyl group that will fit into the S2 subsite of a range of knowncaspases.

(iv) R⁴ is hydrogen or C₁₋₆ alkyl, or R³ and R⁴ taken together form aring of 5-7 ring atoms having 0-2 heteroatoms selected from nitrogen,oxygen or sulfur.

(v) Ring A is a monocyclic, bicyclic or tricyclic heterocyclic orheteroaryl ring system wherein each ring of the system has 5-7 ringatoms.

Ring A is a key feature of compounds of formula Ia. For the Ring Amoiety, bicyclic or tricyclic heterocyclic or heteroaryl rings arepreferred over monocyclic rings. Accordingly, a preferred embodimentrelates to compounds having one or more, and preferably all, of thefollowing features: (i) Z is oxygen; (ii) R¹ is —CH₂OR, —CH₂SR, or—CH₂Y, more preferably R¹ is —CH₂Y, and most preferably, R¹ is —CH₂F;(iii) R² is CO₂H or an ester, amide or isostere thereof; (iv); R³ is agroup having a molecular weight up to about 140 Daltons, such as analiphatic or aralkyl group, more preferably a C₁₋₄ alkyl group; and/or(v) Ring A is a bicyclic or tricyclic heterocyclic or heteroaryl ringsystem wherein each ring of the system has 5-7 ring atoms.

Examples of preferred monocyclic rings for Ring A include triazole,piperidine, morpholine, thiomorpholine, imidazole, pyrrolidine,pyrazole, and piperazine. Examples of preferred bicyclic rings for RingA include indole, isoindole, indoline, indazole, benzimidazole,thieno[3,2-b]pyrrole, dihydroquinoxaline, dihydrocinnoline,dihydronaphthyridine, tetrahydronaphthyridine, tetrahydroquinoline, andtetrahydroisoquinoline, most preferably indole or indoline. Examples ofpreferred tricyclic rings for Ring A include carbazole, phenothiazine,β-carboline, pyrido[4,3-b]indole, 2,3,9-triazafluorene,9-thia-2,10-diazaanthracene, 3,6,9-triazafluorene, phenoxazine,dibenzoazepine, dihydro-dibenzoazepine, dihydrophenazine,dihydroacridine, or dihydrophenanthridine, most preferably carbazole,phenothiazine or dihydrophenanthridine.

Specific examples of compounds I are shown in Table 1.

TABLE 1 Examples of Formula Ia compounds

No. Structure Ia-1

Ia-2

Ia-3

Ia-4

Ia-5

Ia-6

Ia-7

Ia-8

Ia-9

Ia-10

Ia-11

Ia-12

Ia-13

Ia-14

Ia-15

Ia-16

Ia-17

Ia-18

Ia-19

Ia-20

Ia-21

Ia-22

Ia-23

Ia-24

Ia-25

Ia-26

Ia-27

Ia-28

Ia-29

Ia-30

Ia-31

Ia-32

Ia-33

Ia-34

Ia-35

Ia-36

Ia-37

Ia-38

Ia-39

Ia-40

Ia-41

Ia-42

Ia-43

Ia-44

A preferred embodiment of this invention relates to compounds of formulaIa where Ring A is a tricyclic ring system having 1-6 heteroatoms,preferably 1-4 heteroatoms, selected from nitrogen, oxygen or sulfurwherein the end rings of the ring system have 5-7 ring atoms and themiddle ring has 5 or 6 ring atoms. One aspect of this embodiment relatesto compounds of formula II:

where X is a bond, —S—, —O—, —CH₂—, or —NH—, and R¹, R², R³ and R⁴ areas described above. Where X is —CH₂—, each of the methylene hydrogensmay be optionally and independently replaced by —OR, —OH, —SR, protectedOH (such as acyloxy), —CN, —NH₂, —NHR, —N(R)₂, —NHCOR, —NHCONHR,—NHCON(R)₂, —NRCOR, —NHCO₂R, —CO₂R, —CO₂H, —COR, —CONHR, —CON(R)₂,—S(O)₂R, —SONH₂, —S(O)R, —SO₂NHR, —NHS(O)₂R, ═O, ═S, ═NNHR, ═NNR₂,═N—OR, ═NNHCOR, ═NNHCO₂R, ═NNHSO₂R, or ═NR where R is a C₁₋₄ aliphaticgroup. Where X is —NH—, the NH hydrogen may be replaced by alkyl,CO(alkyl), CO₂(alkyl), or SO₂(alkyl). Preferred groups for R¹, R² and R³are as described above.

Another embodiment of this invention relates to compounds of formula Ibthat have one or more, and preferably all, of the following features:

(i) R¹ is hydrogen, —R, —CH₂OR, —CH₂SR, or —CH₂Y. More preferably, R¹ is—CH₂OR, —CH₂SR, or —CH₂Y. An even more preferred R¹ is —CH₂Y. Mostpreferably, R¹ is —CH₂F.

(ii) R² is CO₂H or an ester, amide or isostere thereof.

(iii) R³ is a group having a molecular weight up to about 140 Daltons,such as an aliphatic or aralkyl group. More preferably, R³ is a C₁-C₄alkyl group that fits into the S2 subsite of a range of caspases.

(iv) Ring B is a nitrogen-containing five to seven membered ring having0-1 additional ring heteroatoms selected from nitrogen, oxygen orsulfur.

(v) R⁵ is an optionally substituted C₁₋₆ aliphatic group, an optionallysubstituted phenyl or an optionally substituted benzyl group.

Examples of specific formula Ib compounds are shown below in Table 2.

TABLE 2 Examples of Compounds of Formula Ib Ib-1

Ib-2

Ib-3

Ib-4

The compounds of this invention may be prepared in general by methodsknown to those skilled in the art for analogous compounds, asillustrated by the general scheme below and by the preparative examplesthat follow.

Scheme I above shows a synthetic route for obtaining compounds offormula Ia. Starting compound 1 may be obtained by a variety of generalmethods known in the art for substituted succinic acid derivatives. Forasymmetric approaches to obtain the desired stereochemistry at thechiral centers bearing the R³ and R⁴ groups, see “StereoselectiveAlkylation Reactions of Chiral Metal Enolates” Evans, D. A., inAsymmetric Synthesis, Vol. 3, Chapter 1 pages 1-110; Morrison, J. D.Ed., Academic Press, New York, 1983. In steps (a) and (b) the acidchloride of 1 is formed and then coupled with Ring A as the free amineto provide the amide 2. Step (c) shows a hydrogenolysis of the benzylester to provide carboxylic acid 3. Alternatively, compound 3 may beobtained from other esters using appropriate de-esterificationconditions. In step (d), 3 is coupled with an amino alcohol to providethe amide 4. Depending on the nature of R¹ and R² an amino ketone may beused, in place of the amino alcohol, which avoids the subsequentoxidation step. In the case of fluoromethyl ketones where R¹ is CH₂F,the corresponding amino alcohol may be obtained according to the methodof Revesz et al., Tetrahedron Lett., 1994, 35, 9693. Finally thehydroxyl group in compound 4 is oxidized and the resulting compoundtreated appropriately according to the nature of R². For example, if theproduct Ia requires R² to be a carboxylic acid, then R² in 4 ispreferably an ester and the final step in the scheme is a hydrolysis.

The compounds of this invention are designed to inhibit caspases.Therefore, the compounds of this invention may be assayed for theirability to inhibit apoptosis, the release of IL-1β or caspase activitydirectly. Assays for each of the activities are described below in theTesting section and are also known in the art.

One embodiment of this invention relates to a composition comprising acompound of formula I or a pharmaceutically acceptable salt thereof, anda pharmaceutically acceptable carrier.

If pharmaceutically acceptable salts of the compounds of this inventionare utilized in these compositions, those salts are preferably derivedfrom inorganic or organic acids and bases. Included among such acidsalts are the following: acetate, adipate, alginate, aspartate,benzoate, benzene sulfonate, bisulfate, butyrate, citrate, camphorate,camphor sulfonate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate,pectinate, persulfate, 3-phenyl-propionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate.Base salts include ammonium salts, alkali metal salts, such as sodiumand potassium salts, alkaline earth metal salts, such as calcium andmagnesium salts, salts with organic bases, such as dicyclohexylaminesalts, N-methyl-D-glucamine, and salts with amino acids such asarginine, lysine, and so forth.

Also, the basic nitrogen-containing groups may be quaternized with suchagents as lower alkyl halides, such as methyl, ethyl, propyl, and butylchloride, bromides and iodides; dialkyl sulfates, such as dimethyl,diethyl, dibutyl and diamyl sulfates, long chain halides such as decyl,lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkylhalides, such as benzyl and phenethyl bromides and others. Water oroil-soluble or dispersible products are thereby obtained.

The compounds utilized in the compositions and methods of this inventionmay also be modified by appending appropriate functionalities to enhanceselective biological properties. Such modifications are known in the artand include those which increase biological penetration into a givenbiological system (e.g., blood, lymphatic system, central nervoussystem), increase oral availability, increase solubility to allowadministration by injection, alter metabolism and alter rate ofexcretion.

Pharmaceutically acceptable carriers that may be used in thesecompositions include, but are not limited to, ion exchangers, alumina,aluminum stearate, lecithin, serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

According to a preferred embodiment, the compositions of this inventionare formulated for pharmaceutical administration to a mammal, preferablya human being.

Such pharmaceutical compositions of the present invention may beadministered orally, parenterally, by inhalation spray, topically,rectally, nasally, buccally, vaginally or via an implanted reservoir.The term “parenteral” as used herein includes subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional and intracranial injection orinfusion techniques. Preferably, the compositions are administeredorally or intravenously.

Sterile injectable forms of the compositions of this invention may beaqueous or oleaginous suspension. These suspensions may be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationmay also be a sterile injectable solution or suspension in a non-toxicparenterally acceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilmay be employed including synthetic mono- or di-glycerides. Fatty acids,such as oleic acid and its glyceride derivatives are useful in thepreparation of injectables, as are natural pharmaceutically-acceptableoils, such as olive oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions may alsocontain a long-chain alcohol diluent or dispersant, such ascarboxymethyl cellulose or similar dispersing agents which are commonlyused in the formulation of pharmaceutically acceptable dosage formsincluding emulsions and suspensions. Other commonly used surfactants,such as Tweens, Spans and other emulsifying agents or bioavailabilityenhancers which are commonly used in the manufacture of pharmaceuticallyacceptable solid, liquid, or other dosage forms may also be used for thepurposes of formulation.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, aqueous suspensions or solutions. In thecase of tablets for oral use, carriers that are commonly used includelactose and corn starch. Lubricating agents, such as magnesium stearate,are also typically added. For oral administration in a capsule form,useful diluents include lactose and dried cornstarch. When aqueoussuspensions are required for oral use, the active ingredient is combinedwith emulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Alternatively, the pharmaceutical compositions of this invention may beadministered in the form of suppositories for rectal administration.These may be prepared by mixing the agent with a suitable non-irritatingexcipient which is solid at room temperature but liquid at rectaltemperature and therefore will melt in the rectum to release the drug.Such materials include cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions of this invention may also beadministered topically, especially when the target of treatment includesareas or organs readily accessible by topical application, includingdiseases of the eye, the skin, or the lower intestinal tract. Suitabletopical formulations are readily prepared for each of these areas ororgans.

Topical application for the lower intestinal tract may be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used.

For topical applications, the pharmaceutical compositions may beformulated in a suitable ointment containing the active componentsuspended or dissolved in one or more carriers. Carriers for topicaladministration of the compounds of this invention include, but are notlimited to, mineral oil, liquid petrolatum, white petrolatum, propyleneglycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax andwater. Alternatively, the pharmaceutical compositions may be formulatedin a suitable lotion or cream containing the active components suspendedor dissolved in one or more pharmaceutically acceptable carriers.Suitable carriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutical compositions may be formulated asmicronized suspensions in isotonic, pH adjusted sterile saline, or,preferably, as solutions in isotonic, pH adjusted sterile saline, eitherwith our without a preservative such as benzylalkonium chloride.Alternatively, for ophthalmic uses, the pharmaceutical compositions maybe formulated in an ointment such as petrolatum.

The pharmaceutical compositions of this invention may also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

The above-described compositions are particularly useful in therapeuticapplications relating to an IL-1 mediated disease, an apoptosis mediateddisease, an inflammatory disease, an autoimmune disease, a destructivebone disorder, a proliferative disorder, an infectious disease, adegenerative disease, a disease associated with cell death, an excessdietary alcohol intake disease, a viral mediated disease, uveitis,inflammatory peritonitis, osteoarthritis, pancreatitis, asthma, adultrespiratory distress syndrome, glomerulonephritis, rheumatoid arthritis,systemic lupus erythematosus, scleroderma, chronic thyroiditis, Grave'sdisease, autoimmune gastritis, diabetes, autoimmune hemolytic anemia,autoimmune neutropenia, thrombocytopenia, chronic active hepatitis,myasthenia gravis, inflammatory bowel disease, Crohn's disease,psoriasis, atopic dermatitis, scarring, graft vs host disease, organtransplant rejection, osteoporosis, leukemias and related disorders,myelodysplastic syndrome, multiple myeloma-related bone disorder, acutemyelogenous leukemia, chronic myelogenous leukemia, metastatic melanoma,Kaposi's sarcoma, multiple myeloma, haemorrhagic shock, sepsis, septicshock, burns, Shigellosis, Alzheimer's disease, Parkinson's disease,Huntington's disease, Kennedy's disease, prion disease, cerebralischemia, epilepsy, myocardial ischemia, acute and chronic heartdisease, myocardial infarction, congestive heart failure,atherosclerosis, coronary artery bypass graft, spinal muscular atrophy,amyotrophic lateral sclerosis, multiple sclerosis, HIV-relatedencephalitis, aging, alopecia, neurological damage due to stroke,ulcerative colitis, traumatic brain injury, spinal cord injury,hepatitis-B, hepatitis-C, hepatitis-G, yellow fever, dengue fever, orJapanese encephalitis, various forms of liver disease, renal disease,polyaptic kidney disease, H. pylori-associated gastric and duodenalulcer disease, HIV infection, tuberculosis, and meningitis. Thecompounds and compositions are also useful in treating complicationsassociated with coronary artery bypass grafts and as a component ofimmunotherapy for the treatment of various forms of cancer.

The caspase inhibitors of this invention are also useful in thepreservation of cells, such as tissues and organs. The method of cellpreservation comprises the step of bathing the cells in a solution ofthe compound or a pharmaceutically acceptable derivative thereof.

The amount of compound present in the above-described compositionsshould be sufficient to cause a detectable decrease in the severity ofthe disease or in caspase activity and/or cell apoptosis, as measured byany of the assays described in the examples.

The compounds of this invention are also useful in methods forpreserving cells, such as may be needed for an organ transplant or forpreserving blood products. Similar uses for caspase inhibitors have beenreported (Schierle et al., Nature Medicine, 1999, 5, 97). The methodinvolves treating the cells or tissue to be preserved with a solutioncomprising the caspase inhibitor. The amount of caspase inhibitor neededwill depend on the effectiveness of the inhibitor for the given celltype and the length of time required to preserve the cells fromapoptotic cell death.

According to another embodiment, the compositions of this invention mayfurther comprise another therapeutic agent. Such agents include, but arenot limited to, thrombolytic agents such as tissue plasminogen activatorand streptokinase. When a second agent is used, the second agent may beadministered either as a separate dosage form or as part of a singledosage form with the compounds or compositions of this invention.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of active ingredients will also depend upon the particularcompound and other therapeutic agent, if present, in the composition.

In a preferred embodiment, the invention provides a method of treating amammal, having one of the aforementioned diseases, comprising the stepof administering to said mammal a pharmaceutically acceptablecomposition described above. In this embodiment, if the patient is alsoadministered another therapeutic agent or caspase inhibitor, it may bedelivered together with the compound of this invention in a singledosage form, or, as a separate dosage form. When administered as aseparate dosage form, the other caspase inhibitor or agent may beadministered prior to, at the same time as, or following administrationof a pharmaceutically acceptable composition comprising a compound ofthis invention.

In order that this invention be more fully understood, the followingpreparative and testing examples are set forth. These examples are forthe purpose of illustration only and are not to be construed as limitingthe scope of the invention in any way.

SYNTHESIS EXAMPLES

The following Examples provide synthetic procedures for selectedcompounds of this invention.

Example 1

[3S/R(2S)]3-[2-(Carbazol-9-yl-2-oxo-ethyl)-pentanoylamino]-5-fluoro-4-oxo-pentanoicacid Method A: (4S)-Benzyl-3-pentanoyl-oxazolidin-2-one

A solution of 4(S)-(−)-benzyl-2-oxazolidinone (10 g, 56.43 mmol) inanhydrous THF (200 ml) at −78° C. was treated with a 2.5M solution ofn-butyl lithium in hexanes (23.70 ml, 59.26 mmol) with stirring. Thereaction mixture was allowed to stir at −78° C. for 30 min beforevaleryl chloride (7.57 ml, 62.10 mmol) was added. The reaction mixturewas then allowed to warm to ambient temperature over 15 h after which itwas diluted with NH₄Cl solution, diluted with ethyl acetate and washedwith brine. The organic phase was dried (Na₂SO₄) and concentrated togive a gum. This was purified by flash chromatography (10% EtOAc in40/60 hexanes) to give the sub-title compound (14.61 g, 99%) as acolourless oil: ¹H NMR (400 MHz, CDCl₃) δ 0.94-1.20 (3H, m), 1.35-1.50(2H, m), 1.62-1.80 (2H, m), 2.74-2.84 (1H, m), 2.86-3.08 (2H, m),3.27-3.39 (1H, m), 4.11-4.26 (2H, m), 4.62-4.76 (1H, m), 7.18-7.40 (5H,m).

Method B: [4S(3R)]-3-(4-Benzyl-2-oxo-oxazolidine-3-carbonyl)-hexanoicacid tert-butyl ester

A solution of (4S)-benzyl-3-pentanoyl-oxazolidin-2-one (14.20 g, 54.34mmol) in THF (100 mL) at −78° C. was treated over 10 min with a 1Msolution of sodium bis(trimethylsilyl)amide in THF (59.80 ml, 59.77mmol) with stirring. The reaction mixture was allowed to stir at −78° C.for 30 min before tert-butyl bromoacetate (10.43 ml, 70.64 mmol) wasadded. The reaction mixture was then allowed to stir for a further 3.5 hat −78° C. after which it was diluted with NH₄Cl solution, diluted withethyl acetate and washed sequentially with NaHCO₃ solution and brine.The organic phase was dried (Na₂SO₄) and concentrated to give a gum. Onstanding a white solid formed and this was recrystallized from 40/60DCM/hexanes to give the sub-title compound (14.62 g, 72%) as a whitesolid: ¹H NMR (400 MHz, CDCl₃) δ 0.81-1.20 (3H, m), 1.21-1.76 (13H, m),2.41-2.55 (1H, m), 2.66-2.92 (2H, m), 3.27-3.40 (1H, m), 4.05-4.26 (2H,m), 4.61-4.72 (1H, m), 7.12-7.40 (5H, m).

Method C: (2R)-2-Propyl-succinic acid 1-benzyl ester 4-tert-butyl ester

A solution of benzyl alcohol (4.62 ml, 44.64 mmol) in THF (80 ml) at−20° C. was treated with a 2.5M solution of n-butyl lithium in hexanes(13.36 ml, 33.48 mmol) with stirring. The reaction mixture was allowedto warm to −5° C. over 40 min before a solution of[4S(3R)]-3-(4-benzyl-2-oxo-oxazolidine-3-carbonyl)-hexanoic acidtert-butyl ester (8.38 g, 22.32 mmol) in THF (20 ml) was added. Thereaction mixture was warmed to ambient temperature over 15 h after whichit was diluted with NH₄Cl solution and ethyl acetate and washed withbrine. The organic phase was dried (Na₂SO₄) and concentrated to give agum. This was purified by flash chromatography (11% EtOAc in 40/60hexanes) to give the sub-title compound (4.56 g, 67%) as a colourlessoil: ¹H NMR (400 MHz, CDCl₃) δ 0.83-1.00 (3H, m), 1.21-1.71 (13H, m),2.34-2.45 (1H, m), 2.75-2.95 (1H, m), 5.09-5.25 (2H, m), 7.30-7.43 (5H,m).

Method D: (2R)-2-Propyl-succinic acid 1-benzyl ester

A stirred solution of (2R)-2-propyl-succinic acid 1-benzyl ester4-tert-butyl ester (4.56 g, 14.88 mmol) in anhydrous DCM (20 mL), at 0°C., was treated with a solution of trifluoroacetic acid (10 mL) inanhydrous DCM (10 mL). The reaction mixture was allowed to warm toambient temperature over 3 h before being concentrated under reducedpressure. The residue was dissolved in dry DCM, before concentratingagain. This process was repeated several times in order to remove excesstrifluoroacetic acid to leave the sub-title compound (3.70 g, 99%) as agum: ¹H NMR (400 MHz, CDCl₃) δ 0.82-0.99 (3H, m), 1.21-1.76 (4H, m),2.45-2.60 (1H, m), 2.76-3.00 (2H, m), 5.10-5.21 (2H, m), 7.28-7.43 (5H,m), 7.83-8.18 (1H, m).

Method E: (2R)-2-(2-Carbazol-9-yl-2-oxo-ethyl)-pentanoic acid benzylester

A stirred solution of carbazole (2.49 g, 14.88 mmol) in anhydrous THF(30 mL), at −78° C., was treated with a 1.0M solution of lithiumbis(trimethylsilyl)amide in THF (14.88 ml, 14.88 mmol). The reactionmixture was allowed to warm to ambient temperature over 2 h before beingre-cooled to −78° C.

A solution of (2R)-2-Propyl-succinic acid 1-benzyl ester (3.70 g, 14.78mmol) in anhydrous DCM (20 mL), stirring at 0° C., was treated withoxalyl chloride (1.43 ml, 16.37 mmol) and DMF (14 drops). The reactionmixture was stirred at 0° C. for 1 h before being concentrated in vacuo.The residue was dissolved in anhydrous THF (10 ml) and added to thelithium anion of carbazole previously prepared, at −78° C. The reactionmixture was warmed to ambient temperature over 40 h after which it wasdiluted with NH₄Cl solution, and ethyl acetate and washed sequentiallywith 2N HCl, NaHCO₃ solution and brine. The organic phase was dried(Na₂SO₄) and concentrated to give a gum which was purified by flashchromatography (10% EtOAc in 40/60 hexanes) to give the sub-titlecompound (4.50 g, 76%) as a semi solid/oil which also containedcarbazole: ¹H NMR (400 MHz, CDCl₃) δ 0.82-1.05 (3H, m), 1.11-1.99 (4H,m), 3.18-3.38 (2H, m), 3.56-3.71 (1H, m), 5.10-5.30 (2H, m), 7.11-7.60(9H, m), 7.92-8.29 (4H, m).

Method F: (2R)-2-(2-Carbazol-9-yl-2-oxo-ethyl)-pentanoic acid

A stirred solution of (2R)-2-(2-Carbazol-9-yl-2-oxo-ethyl)-pentanoicacid benzyl ester (4.50 g, 11.26 mmol) in EtOAc (60 mL) was treated with10% Pd on carbon (˜400 mg) and the reaction mixture then placed under anatmosphere of hydrogen. After 1 h further 10% Pd on carbon (˜300 mg) wasadded and the reaction mixture was placed under hydrogen, with stirring,for a further 3 h after which the reaction mixture was filtered througha celite pad and concentrated to give the sub-title compound (2.94 g,84%) as a white solid which also contained carbazole: ¹H NMR (400 MHz,CDCl₃) δ 0.92-1.04 (3H, m), 1.32-2.00 (4H, m), 3.19-3.34 (2H, m),3.58-3.70 (1H, m), 7.30-7.53 (4H, m), 8.00-8.30 (4H, m).

Method G: [3S/R, 4S/R,(2R)]-3-[2-(2-Carbazol-9-yl-2-oxo-ethyl)-pentanoylamino]-5-fluoro-4-hydroxy-pentanoicacid tert-butyl ester

A stirred mixture of (2R)-2-(2-carbazol-9-yl-2-oxo-ethyl)-pentanoic acid(2.94 g, 9.50 mmol), 3-amino-5-fluoro-4-hydroxy-pentanoic acidtert-butyl ester (2.07 g, 9.99 mmol), HOBT (1.41 g, 10.43 mmol), DMAP(1.34 g, 10.97 mmol) and anhydrous THF (40 mL) was cooled to 0° C. thenEDC (2.00 g, 10.43 mmol) was added. The mixture was allowed to warm toroom temperature during 16 h then concentrated under reduced pressure.The residue purified by flash chromatography (33% EtOAc in 40/60hexanes) to give the sub-title compound (2.51 g, 53%) as a foam: ¹H NMR(400 MHz, CDCl₃) δ 0.90-1.03 (3H, m), 1.20-1.90 (13H, m), 2.50-3.00 (3H,m), 3.12-3.26 (1H, m), 3.59-3.80 (2H, m), 4.00-4.68 (3H, m), 6.53-6.89(1H, m), 7.30-7.52 (4H, m), 7.95-8.05 (2H, m), 8.15-8.26 (2H, m) ¹⁹F NMR(376 MHz, CDCl₃) δ −229.10, −229.34, −230.95, −231.09.

Method H: [3S/R,(2R)]-3-[2-(2-Carbazol-9-yl-2-oxo-ethyl)-pentanoylamino]-5-fluoro-4-oxo-pentanoicacid tert-butyl ester

A stirred solution of [3S/R, 4S/R,(2R)]-3-[2-(2-carbazol-9-yl-2-oxo-ethyl)-pentanoylamino]-5-fluoro-4-hydroxy-pentanoicacid tert-butyl ester (2.51 g, 5.03 mmol) in anhydrous DCM (60 ml) wastreated with 1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3(1H)-one(2.35 g, 5.53 mmol) at 0° C. The resulting mixture was kept at 0° C. for3 h, diluted with DCM, and then washed sequentially with saturatedaqueous sodium thiosulphate, NaHCO₃ solution and brine. The organicswere dried (Na₂SO₄) and concentrated. The residue was purified by flashchromatography (25% ethyl acetate in 40/60 hexanes) to afford thesub-title compound as an off white solid (1.437 g, 57%): IR (solid)1722, 1689, 1636, 1531, 1441, 1365, 1279, 1155 cm⁻¹; ¹H NMR (400 MHz,CDCl₃) δ 0.85-1.50 (3H, m), 1.35-1.54 (11H, m), 1.55-1.69 (1H, m),1.78-1.95 (1H, m), 2.67-3.28 (4H, m), 3.60-3.79 (1H, m), 4.80-5.59 (3H,m), 6.89-7.04 (1H, m), 7.33-7.54 (4H, m), 7.98-8.04 (2H, m), 8.15-8.28(2H, m); ¹³C (100 MHz, CDCl₃) δ 14.12, 14.40, 14.47, 14.60, 20.78,20.84, 21.47, 28.32, 28.42, 28.48, 29.77, 33.63, 34.58, 34.91, 40.05,43.05, 43.26, 43.29, 52.60, 53.00, 53.64, 66.90, 66.99, 82.62, 82.69,85.53, 116.88, 116.94, 120.28, 120.31, 124.27, 127.76, 127.86, 128.69,128.77, 128.99, 138.80, 171.21, 171.29, 172.21, 172.25, 175.53, 176.03,203.04, 203.20, 203.30, 203.46; ¹⁹F (376 MHz, CDCl₃) δ −232.12, −233.24.

Method I:[3S/R,(2R)]-3-[2-(2-Carbazol-9-yl-2-oxo-ethyl)-pentanoylamino]-5-fluoro-4-oxo-pentanoicacid

A solution of[3S/R,(2R)]-3-[2-(2-carbazol-9-yl-2-oxo-ethyl)-pentanoylamino]-5-fluoro-4-oxo-pentanoicacid tert-butyl ester (1.43 g, 2.88 mmol) in anhydrous DCM (20 ml) wastreated with a solution of TFA (10 ml) in anhydrous DCM (10 ml) withstirring. The mixture was stirred at 0° C. for 2 h then at roomtemperature for 2 h. The mixture was concentrated under reduced pressureand then the residue was dissolved in dry DCM. This process was repeatedseveral times in order to remove excess trifluoroacetic acid. Theoff-white solid was recrystallized from Et₂O/40/60 hexanes to give thetitle compound as a white powder (71 mg): IR (solid) 1746, 1689, 1641,1541, 1436, 1374, 1284, 1207, 1160 cm⁻¹; ¹H NMR (400 MHz, d₆-DMSO) δ0.80-1.00 (3H, m), 1.20-1.76 (4H, m), 2.30-2.90 (2H, m), 2.95-3.24 (1H,m), 3.26-3.59 (2H, m), 4.25-4.79 (1.5H, m), 5.02-5.43 (1.5H, m),7.36-7.58 (4H, m), 8.10-8.30 (4H, m), 8.54-8.91 (1H, m); ¹³C NMR (100MHz, DMSO) δ 14.31, 20.03, 20.13, 21.92, 22.51, 34.36, 34.77, 41.20,41.62, 44.06, 51.77, 52.84, 83.45, 85.22, 116.70, 120.54, 123.91,124.01, 127.85, 126.01, 138.20, 172.15, 172.36, 172.96, 173.00, 175.32,175.48, 202.60, 203.10; ¹⁹F (376 MHz, DMSO) δ −226.68, −226.73, −231.21,−232.95, −233.38, −233.52.

Example 2

[3S/R(2S)]-3-[2-(2-Carbazol-9-yl-2-oxo-ethyl)-3-methyl-butyrylamino]-5-fluoro-4-oxo-pentanoicacid

This was prepared using procedures similar to those described in MethodsA-I. The product was isolated as a white powder (71% for final step): IR(solid) 1739, 1682, 1646, 1545, 1447, 1381, 1290, 1209, 1170 cm⁻¹; ¹HNMR (400 MHz, DMSO+TFA) δ 0.79-1.08 (6H, m), 1.89-2.15 (1H, m),2.31-3.60 (5H, m), 4.21-4.78 (1.25H, m), 4.98-5.45 (1.75H, m), 7.38-7.60(4H, m), 8.14-8.35 (4H, m), 8.56-8.90 (1H, m); ¹³NMR (100 MHz, DMSO) δ20.46, 20.84, 21.04, 21.21, 30.77, 30.85, 33.37, 34.83, 35.24, 38.16,38.89, 47.67, 48.23, 52.19, 53.43, 83.96, 84.01, 85.72, 85.77, 117.16,121.02, 124.43, 126.42, 126.52, 128.42, 138.75, 172.64, 172.90, 173.85,173.90, 174.74, 174.93, 175.16, 202.91, 203.04, 203.51, 203.65; ¹⁹F (376MHz, DMSO) δ −226.63, −226.68, −231.24, −233.16, −233.38, −233.55.

Testing Methods

Enzyme Assays

The assays for caspase inhibition are based on the cleavage of afluorogenic substrate by recombinant, purified human Caspases-1, -3, -7or -8. The assays are run in essentially the same way as those reportedby Garcia-Calvo et al. (J. Biol. Chem. 273 (1998), 32608-32613), using asubstrate specific for each enzyme. The substrate for Caspase-1 isAcetyl-Tyr-Val-Ala-Asp-amino-4-methylcoumarin. The substrate forCaspases-3, -7 and -8 is Acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin.The observed rate of enzyme inactivation at a particular inhibitorconcentration, k_(obs), is computed by direct fits of the data to theequation derived by Thornberry et al. (Biochemistry 33 (1994),3943-3939) using a nonlinear least-squares analysis computer program(PRISM 2.0; GraphPad software). To obtain the second order rateconstant, k_(inact), k_(obs) values are plotted against their respectiveinhibitor concentrations and k_(inact) values are subsequentlycalculated by computerized linear regression. Many of the presentcompounds that were tested showed the following activities: againstcaspase-1, k_(inact) values between 25,000 and 1,500,000 M⁻¹s⁻¹; againstcaspase-3, k_(inact) values between 9,000 and 1,500,000 M⁻¹s⁻¹; againstcaspase-8, k_(inact) values between 10,000 and 700,000 M⁻¹s⁻¹.

Inhibition of IL-1β Secretion from Mixed Population of Peripheral BloodMononuclear Cells (PBMC)

Processing of pre-IL-1β by caspase-1 may be measured in cell cultureusing a variety of cell sources. Human PBMC obtained from healthy donorsprovides a mixed population of lymphocyte and mononuclear cells thatproduce a spectrum of interleukins and cytokines in response to manyclasses of physiological stimulators.

Experimental Procedure

The test compound is dissolved in dimethyl sulfoxide (DMSO, Sigma#D-2650) to give a 100 mM stock solution. This is diluted in completemedium consisting of RPMI containing 10% heat inactivated FCS (Gibco BRL#10099-141), 2 mM L-Glutamine (Sigma, #G-7513), 100 U penicillin and 100μg/ml streptomycin (Sigma #P-7539). The final concentration range oftest compound is from 100 μM down to 6 nM over eight dilution steps. Thehighest concentration of test compound is equivalent to 0.1% DMSO in theassay.

Human PBMC are isolated from Buffy Coats obtained from the blood bankusing centrifugation on Ficoll-Paque leukocyte separation medium(Amersham, #17-1440-02) and the cellular assay is performed in a sterile96 well flat-bottomed plate (Nunc). Each well contains 100 μl of thecell suspension, 1×10⁵ cells, 50 μl of compound dilutions and 50 μl ofLPS (Sigma #L-3012) at 50 ng/ml final concentration. Controls consist ofcells+/−LPS stimulation and a serial dilution of DMSO diluted in thesame way as compound. The plates are incubated for 16-18 h at 37° C. in5% CO₂ & 95% humidity atmosphere.

After 16-18 h the supernatants are harvested after centrifuging theplates at 100×g at 18° C. for 15 min and assayed for their IL-1βcontent. Measurement of mature IL-1β in the supernatant is performedusing the Quantikine kits (R&D Systems) according to manufacturer'sinstructions. Mature IL-1β levels of about 600-1500 pg/ml are observedfor PBMCs in positive control wells.

The inhibitory potency of the compounds may be represented by an IC₅₀value, which is the concentration of inhibitor at which 50% of themature IL-1β is detected in the supernatant as compared to the positivecontrols.

Table 5 shows inhibition of IL-1β secretion from peripheral bloodmononuclear cells for selected compounds of this invention as determinedby the above methods.

Selected compounds have been tested in this assay and shown to inhibitIL-1β release with IC₅₀ values between 0.04 μM and 20 μM.

Anti-Fas Induced Apoptosis Assay

Cellular apoptosis may be induced by the binding of Fas ligand (FasL) toits receptor, CD95 (Fas). CD95 is one of a family of related receptors,known as death receptors, which can trigger apoptosis in cells viaactivation of the caspase enzyme cascade. The process is initiated bythe binding of the adapter molecule FADD/MORT-1 to the cytoplasmicdomain of the CD-95 receptor-ligand complex. Caspase-8 then binds FADDand becomes activated, initiating a cascade of events that involve theactivation of downstream caspases and subsequent cellular apoptosis.Apoptosis can also be induced in cells expressing CD95 eg the JurkatE6.1 T cell lymphoma cell line, using an antibody, rather than FasL, tocrosslink the cell surface CD95. Anti-Fas-induced apoptosis is alsotriggered via the activation of caspase-8. This provides the basis of acell based assay to screen compounds for inhibition of thecaspase-8-mediated apoptotic pathway.

Experimental Procedure

Jurkat E6.1 cells are cultured in complete medium consisting ofRPMI-1640 (Sigma No)+10% foetal calf serum (Gibco BRL No. 10099-141)+2mM L-glutamine (Sigma No. G-7513). The cells are harvested in log phaseof growth. 100 ml of cells at 5-8×10⁵ cells/ml are transferred tosterile 50 ml Falcon centrifuge tubes and centrifuged for 5 minutes at100×g at room temperature. The supernatant is removed and the combinedcell pellets resuspended in 25 ml of complete medium. The cells arecounted and the density adjusted to 2×10⁶ cells/ml with complete medium.

The test compound is dissolved in dimethyl sulfoxide (DMSO) (Sigma No.D-2650) to give a 100 mM stock solution. This is diluted to 400 μm incomplete medium, then serially diluted in a 96-well plate prior toaddition to the cell assay plate.

100 μl of the cell suspension (2×10⁶ cells) is added to each well of asterile 96-well round-bottomed cluster plate (Costar No. 3790). 50 μl ofcompound solution at the appropriate dilution and 50 μl of anti-Fasantibody, clone CH-11 (Kamiya No. MC-060) at a final concentration of 10ng/ml, are added to the wells. Control wells are set up minus antibodyand minus compound but with a serial dilution of DMSO as vehiclecontrol. The plates are incubated for 16-18 hrs at 37° C. in 5% CO₂ and95% humidity.

Apoptosis of the cells is measured by the quantitation of DNAfragmentation using a ‘Cell Death Detection Assay’ fromBoehringer-Mannheim, No. 1544 675. After incubation for 16-18 hrs theassay plates are centrifuged at 100×g at room temperature for 5 minutes.150 μl of the supernatant are removed and replaced by 150 μl of freshcomplete medium. The cells are then harvested and 200 μl of the lysisbuffer supplied in the assay kit are added to each well. The cells aretriturated to ensure complete lysis and incubated for 30 minutes at 4°C. The plates are then centrifuged at 1900×g for 10 minutes and thesupernatants diluted 1:20 in the incubation buffer provided. 100 μl ofthis solution is then assayed according to the manufacturer'sinstructions supplied with the kit. OD₄₀₅ nm is measured 20 minutesafter addition of the final substrate in a SPECTRAmax Plus plate reader(Molecular Devices). OD405 nm is plotted versus compound concentrationand the IC50 values for the compounds are calculated using thecurve-fitting program SOFTmax Pro (Molecular Devices) using the fourparameter fit option.

Selected compounds have been tested in this assay and shown to inhibitFas-induced apoptosis of Jurkat cells with IC₅₀ values between 0.001 μMand 0.15 μM.

While we have described a number of embodiments of this invention, it isapparent that our basic examples may be altered to provide otherembodiments, which utilize the compounds and methods of this invention.Therefore, it will be appreciated that the scope of this invention is tobe defined by the appended claims rather than by the specificembodiments, which have been represented by way of example.

1. A compound of formula Ib:

wherein:

 between R³ and R⁴ represents a single or double bond; Z is oxygen orsulfur; R¹ is hydrogen, —CHN₂, —R, —CH₂OR, —CH₂SR, or —CH₂Y; R is aC₁₋₁₂ aliphatic, aryl, aralkyl, heterocyclyl, or heterocyclylalkyl; Y isan electronegative leaving group; R² is CO₂H, CH₂CO₂H, or esters, amidesor isosteres thereof; R³ is hydrogen, a side chain of a natural α-aminoacid, or a substituted or unsubstituted group having a molecular weightup to about 140 Daltons selected from C₁₋₁₂ aliphatic, 5-14 memberedaryl, (5-14 membered aryl) -(C₁₋₁₂-alkyl)-, 3-9 membered heterocyclyl,or (3-9 membered heterocyclyl)-(C₁₋₁₂ alkyl)-; wherein said R³ isoptionally substituted with halogen, —R, —OR, —OH, —SH, —SR, acyloxy,phenyl (Ph), —OPh, —NO₂, —CN, —NH₂, —NHR, —N(R)₂, —NHCOR, —NHCONHR,—NHCON(R)₂, —NRCOR, —NHCO₂R, —CO₂R, —CO₂H, —COR, —CONHR, —CON(R)₂,—S(O)₂R, —SONH₂, —S(O)R, —SO₂NHR, —NHS(O)₂R, ═O, ═S, ═NNHR, ═NNR₂,═N—OR, ═NNHCOR, ═NNHCO₂R, ═NNHSO₂R, or ═NR; R is C₁₋₆ aliphatic; Ring Bis a nitrogen-containing 5-7 membered ring having 0-2 additional ringheteroatoms selected from nitrogen, oxygen or sulfur; R⁵ is R⁶,(CH₂)_(n)R⁶, COR⁶, CO₂R⁶, SO₂R⁶, CON(R⁶)₂, or SO₂N(R⁶)₂; n is one tothree; and each R⁶ is independently selected from hydrogen, anoptionally substituted C₁₋₄ aliphatic group, an optionally substitutedC₆₋₁₀ aryl group, or a mono- or bicyclic heteroaryl group having 5-10ring atoms.
 2. The compound of claim 1, wherein Z is oxygen.
 3. Thecompound of claim 2, as represented by the formula shown below:


4. The compound of claim 1, wherein R³ is a C₁-C₄ alkyl group.
 5. Thecompound of claim 1, wherein the compound has one or more of thefollowing features: (i) R¹ is —CH₂OR, —CH₂SR, or —CH₂Y; (ii) R² is CO₂Hor an ester, amide or isostere thereof; (iii) R³ is hydrogen, a sidechain of a natural α-amino acid, or a substituted or unsubstituted grouphaving a molecular weight up to about 140 Daltons selected from C₁₋₁₂aliphatic, 5-14 membered aryl, (5-14 membered aryl)-(C₁₋₁₂-alkyl)-, 3-9membered heterocyclyl, or (3-9 membered heterocyclyl)-(C₁₋₁₂ alkyl)-;wherein said R³ is optionally substituted with halogen, —R, —OR, —OH,—SH, —SR, acyloxy, phenyl (Ph), —OPh, —NO₂, —CN, —NH₂, —NHR, —N(R)₂,—NHCOR, —NHCONHR, —NHCON(R)₂, —NRCOR, —NHCO₂R, —CO₂R, —CO₂H, —COR,—CONHR, —CON(R)₂, —S(O)₂R, —SONH₂, —S(O)R, —SO₂NHR, —NHS(O)₂R, ═O, ═S,═NNHR, ═NNR₂, ═N—OR, ═NNHCOR, ═NNHCO₂R, ═NNHSO₂R, or ═NR; R is C₁₋₆aliphatic; (iv) Ring B is a nitrogen-containing five to seven memberedring having 0-1 additional ring heteroatoms selected from nitrogen,oxygen or sulfur; and (v) R⁵ is an optionally substituted C₁₋₆ aliphaticgroup, an optionally substituted phenyl or an optionally substitutedbenzyl group.
 6. The compound of claim 1, wherein the compound has thefollowing features: (i) R^(l) is —CH₂OR, —CH₂SR, or —CH₂Y; (ii) R² isCO₂H or an ester, amide or isostere thereof; (iii) R³ is hydrogen, aside chain of a natural α-amino acid, or a substituted or unsubstitutedgroup having a molecular weight up to about 140 Daltons selected fromC₁₋₁₂ aliphatic, 5-14 membered aryl, (5-14 memberedaryl)-(C₁₋₁₂-alkyl)-, 3-9 membered heterocyclyl, or (3-9 memberedheterocyclyl)-(C₁ ₋₁₂alkyl)-; wherein said R³ is optionally substitutedwith halogen, —R, —OR, —OH, —SH, —SR, acyloxy, phenyl (Ph), —OPh, —NO₂,—CN, —NH₂, —NHR, —N(R)₂, —NHCOR, —NHCONHR, —NHCON(R)₂, —NRCOR, —NHCO₂R,—CO₂R, —CO₂H, —COR, —CONHR, —CON(R)₂, —S(O)₂R, —SONH₂, —S(O)R, —SO₂NHR,—NHS(O)₂R, ═O, ═S, ═NNHR, ═NNR₂, ═N—OR, ═NNHCOR, ═NNHCO₂R, ═NNHSO₂R, or═NR; R is C₁₋₆ aliphatic; (iv) Ring B is a nitrogen-containing five toseven membered ring having 0-1 additional ring heteroatoms selected fromnitrogen, oxygen or sulfur; and (v) R⁵ is an optionally substituted C₁₋₆aliphatic group, an optionally substituted phenyl or an optionallysubstituted benzyl group.
 7. The compound of claim 6, wherein R^(l) is—CH₂Y.
 8. The compound of claim 7, wherein R^(l) is —CH₂F.
 9. Thecompound of claim 8, wherein R³ is a C₁₋₄ alkyl group.
 10. The compoundof claim 1, selected from the following: